Staphylococcus aureus (SA) is a major human pathogen that can cause life threatening systemic infections. In fact, S. aureus is the most common cause of nosocomial bacteremia with a prevalence of 26%. Despite improvements in clinical care, SA bloodstream infection (BSI) continues to pose a major challenge due to high rate of treatment failure and mortality. Infections that are caused by methicillin-resistant SA (MRSA) strains are becoming increasingly resistant to antibiotics, leading to treatment failures and poor patient outcomes. If host- directed therapies such an effective SA vaccine can provide an alternative to antibiotics, a greater understanding of immune mechanisms that promote protection is essential. This is especially important since all SA vaccination attempts over the past two decades have failed in human trials. Notably, in a recent trial, a vaccine targeting SA iron surface determinant B (IsdB) against SA infections following cardiothoracic surgery had the opposite effect and patients who were vaccinated and suffered a SA infection had a 2-fold higher rate of multiorgan failure and a 5-fold higher mortality than placebo controls. While reasons for this catastrophic outcome are still unknown, a post-hoc study suggested that absent or low serum T cell cytokine levels (IL-2 and IL-17) at the time of vaccination predisposed them for treatment failure. There is therefore an urgent need to understand the underlying immunological mechanisms that lead to these deleterious outcomes. Our preliminary findings in mice recapitulate deleterious immune responses triggered by immunizing mice with a lethally irradiated whole cell vaccine or by intradermal exposure to SA that led to exacerbated disease and mortality upon SA BSI challenge. This phenotype was associated with a CD4 T cell mediated, IFN?-dependent immunopathology and likely an imbalance of Th1/Th17 responses. The goal of this proposal is to test the overarching hypothesis that prior exposure to SA antigens and virulence factors or cutaneous SA infection impact natural- and vaccine-elicited CD4 T cell responses during a subsequent SA BSI. To this end, using mice we will fully characterize the T cell responses to two vaccine platforms and will evaluate how the initial response to SA surface antigens or to a toxoid vaccine differentially regulate the CD4 T cell response, and consequently the vaccine-elicited immunity against subsequent SA BSI and if these responses can be influenced by different vaccine adjuvants (Aim 1). Using WT mice and human HLA-DR4 transgenic mice as well as several WT and isogenic mutant SA strains we will investigate how pore forming toxins and superantigens modulate CD4 T cell responses that affect natural and vaccine induced immunity against SA BSI (Aim 2). We will also establish and use a novel nonhuman primate model of SA BSI and evaluate the impact of pre-exposure on vaccine immunogenicity, with a focus on CD4 T cell responses, and efficacy of our multivalent toxoid vaccine (Aim 3). The ultimate goal of this proposal is to understand protective vaccine-induced immunity against SA BSI in complementary pre-clinical models to deepen our understanding of immunity to SA BSI to help guide more effective vaccines to prevent disastrous outcomes in future clinical trials.